Response of Starved Rats and Polycythemic Rats to Graded Doses of Erythropoietin. ∗

Abstract

Present studies of the humoral regulation of erythropoiesis are based on diverse in vivo bio-assay technics, described by Gordon(1). Gordon also noted that methodology influenced results. Few specific attempts have been made to compare results obtained by different assay procedures(2,3).

This report details results of assays carried out using starved rats or rats rendered polycythemic by hypertransfusion. Reticulocyte counts and red cell Fe³⁹ incorporation were measured; erythropoietic response of both types of assay animal to human and rabbit erythropoietin was assessed. Serially graded doses of erythropoietin were assayed and the relationship between response in red cell iron incorporation or reticulocyte production and dose of erythropoietin was determined.

Methods and materials. Erythropoietin was prepared from rabbit plasma and human urine: a) Hemolytic anemia was produced in albino rabbits by daily subcutaneous injections of 1 ml of 2.5% phenylhydrazine solution. When the hematocrits were 15% or less the rabbits were exsanguinated into heparinized flasks. The separated plasma was pooled, and extracted by the method of Borsook(4). This extract was concentrated 10-fold, dialyzed for 12 hours against distilled water at 4°C and stored frozen. The erythropoietic stimulating activity of such extracts had been previously demonstrated(4,5). b) Urine was obtained from a patient with severe anemia due to erythroid failure. His hematocrit ranged between 17 and 20%; there was persistent, absolute reticulocytopenia, and marrow was devoid of red cell precursors. Urine was dialyzed against running tap water for 18 hours, concentrated by flash evaporation to one-twentieth of the original volume and stored at −18°C.

Two types of bio-assay were used. On the first day of a 4-day assay period, female Sprague-Dawley rats weighing 180 to 200 g were started on starvation(6) or rendered polycythemic by a single transfusion of homologous packed red cells equivalent to 50% of the animals' original predicted red cell mass(7).